Contribution of an SFK-Mediated Signaling Pathway in the Dorsal Hippocampus to Cocaine-Memory Reconsolidation in Rats

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1 2 3 3 4 4 Audrey M Wells , Xiaohu Xie , Jessica A Higginbotham , Amy A Arguello , Kati L Healey , Megan Blanton

and Rita A Fuchs*,3

1 2 Behavioral Genetics Laboratory, Department of Psychiatry, Harvard Medical School, McLean Hospital, Belmont, CA, USA; Department of 3 Chemistry, North Carolina State University, Raleigh, NC, USA; Department of Integrative Physiology and Neuroscience, Washington State

University College of Veterinary Medicine, Pullman, WA, USA; 4Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill,

NC, USA

– – Environmentally induced relapse to cocaine seeking requires the retrieval of context response cocaine associative memories. These

memories become labile when retrieved and must undergo reconsolidation into long-term memory storage to be maintained. Identification

of the molecular underpinnings of cocaine-memory reconsolidation will likely facilitate the development of treatments that mitigate the

impact of cocaine memories on relapse vulnerability. Here, we used the rat extinction-reinstatement procedure to test the hypothesis that

the Src family of tyrosine kinases (SFK) in the dorsal hippocampus (DH) critically controls contextual cocaine-memory reconsolidation. To

μ this end, we evaluated the effects of bilateral intra-DH microinfusions of the SFK inhibitor, PP2 (62.5 ng per 0.5 l per hemisphere),

following re-exposure to a cocaine-associated (cocaine-memory reactivation) or an unpaired context (no memory reactivation) on

subsequent drug context-induced instrumental cocaine-seeking behavior. We also assessed alterations in the phosphorylation state of SFK

targets, including GluN2A and GluN2B N-methyl-D-aspartate (NMDA) and GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid receptor subunits at the putative time of memory restabilization and following PP2 treatment. Finally, we evaluated the effects of intra-

DH PEAQX (2.5 μg per 0.5 μl per hemisphere), a GluN2A-subunit-selective NMDAR antagonist, following, or in the absence of, cocaine-

memory reactivation on subsequent drug context-induced cocaine-seeking behavior. GluN2A phosphorylation increased in the DH during

— — putative memory restabilization, and intra-DH PP2 treatment inhibited this effect. Furthermore, PP2 as well as PEAQX attenuated

subsequent drug context-induced cocaine-seeking behavior, in a memory reactivation-dependent manner, relative to VEH. These findings

suggest that hippocampal SFKs contribute to the long-term stability of cocaine-related memories that underlie contextual stimulus control

over cocaine-seeking behavior.

Neuropsychopharmacology (2016) 41, 675–685; doi:10.1038/npp.2015.217; published online 12 August 2015

INTRODUCTION cocaine-related memories can be therapeutically modified by interfering with memory reconsolidation (Tronson and Cocaine-associated environments evoke craving and Taylor, 2013). promote relapse, thereby impeding the successful treatment The dorsal hippocampus (DH) regulates the reconsolida- of cocaine addiction (Gawin and Kleber, 1986; Childress tion of contextual cocaine memories that direct instrumental et al, 1988). Implicit in this is the retrieval and utilization of – – cocaine-seeking behavior (Ramirez et al, 2009) through context response cocaine memories (Sorg, 2012; Tronson interactions with the basolateral amygdala (BLA; Wells et al, and Taylor, 2013). It is theorized that associative memories 2011). However, unlike the BLA, the DH contributes to are restabilized into long-term memory (LTM) storage via a cocaine-memory reconsolidation in a protein synthesis- protein synthesis-dependent memory reconsolidation independent manner that is poorly understood (Ramirez process following their retrieval and destabilization (Nader et al, 2009). Characterizing the intracellular signaling path- et al, 2000a). Therefore, it is possible that destabilized ways in the DH that control cocaine-memory reconsolidation fills a gap in our understanding and may advance the *Correspondence: Dr RA Fuchs, Department of Integrative Physiology development of novel therapeutics for relapse prevention. and Neuroscience, Washington State University College of Veterinary Medicine, PO Box 647620, Pullman, WA 99164-7620, USA, N-methyl-D-aspartate receptor (NMDAR) stimulation is Tel: +1 509 335 6164, Fax: +1 509 335 4650, necessary for drug-memory reconsolidation in several E-mail: [email protected] experimental procedures (Kelley et al, 2007; Sadler et al, Received 28 January 2015; revised 13 July 2015; accepted 15 July 2015; 2007; Brown et al, 2008; Milton et al, 2008a). NMDAR accepted article preview online 23 July 2015 function is regulated by the Src family of tyrosine kinases SFKs in cocaine-memory reconsolidation AM Wells et al 676 (SFKs; Salter and Kalia, 2004), which includes Src, Fyn, Lyn, Self-Administration and Extinction Training Yes, and Lck (Ohinishi et al, 2011). SFKs phosphorylate Rats were randomly assigned to one of two distinctly GluN2A (Nakazawa et al, 2001; Taniguchi et al, 2009) and different contexts (Context A or B, see Supplementary GluN2B NMDAR subunits (Nakazawa et al, 2001; Table S1) for self-administration training, which took place Prybylowski et al, 2005; Stramiello and Wagner, 2008) to during daily, 2 h sessions during the dark cycle. During these promote synaptic plasticity and memory (Purcell and Carew, 2003; Ohinishi et al, 2011). Specifically, SFK-mediated sessions, responses on a designated active lever produced cocaine infusions (cocaine hydrochloride; 0.15 mg per phosphorylation of GluN2A subunits is associated with 0.05 ml per infusion, intravenously; NIDA, Research Trian- synaptic strengthening and long-term potentiation (LTP; gle Park, NC) under an FR1 − 20 s time-out reinforcement Yang et al, 2012). Additionally, SFK activation in the dorsal schedule, as described previously (Fuchs et al, 2009). Presses striatum and DH is necessary for drug-induced behavioral on a second, inactive lever were recorded but had no phenomena, such as drug-primed ethanol-seeking (Wang scheduled consequences. Training continued until rats met et al, 2010) and context-elicited cocaine-seeking behaviors the acquisition criterion (ie, ⩾ 10 sessions with ⩾ 10 cocaine (Xie et al, 2013), respectively. However, the role of SFKs in infusions per session). drug-memory reconsolidation is unknown. Next, rats received seven daily 2 h extinction-training The present study tested the hypothesis that SFK-mediated signaling in the DH is necessary for the reconsolidation of sessions in the other context. Lever presses were recorded but had no programmed consequences. Rats in Experiments 1 contextual cocaine memories that promote instrumental and 3 were adapted to the intracranial microinfusion cocaine-seeking behavior. In Experiment 1, we evaluated (a) the effects of SFK inhibition in the DH during putative procedure after session 4. Rats in Experiment 2 were adapted to the same procedure after sessions 6 and 7 to minimize the memory reconsolidation on subsequent drug context-induced potential effects of infusion stress on protein expression (see cocaine-seeking behavior and (b) whether these effects Supplementary Methods). depended on explicit memory reactivation. In Experiment 2, we used quantitative western blotting to determine whether SFK inhibition in the DH during cocaine-memory reconsoli- Experiment 1a dation attenuates GluN2A NMDAR subunit phosphorylation. We also evaluated changes in alternate targets of SFKs: GluN2B In Experiment 1a, we evaluated whether intra-DH NMDA and GluA2 AMPA (α-amino-3-hydroxy-5-methyl-4- microinfusions of 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)- isoxazolepropionic acid) receptor subunits (Hayashi et al, 1999; 1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophe- Hayashi and Huganir, 2004). Finally, in Experiment 3, we nyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), an ATP- assessed the effects of a GluN2A-preferring NMDAR antago- competitive inhibitor of SFKs (Hanke et al, 1996), adminis- nist in the DH on cocaine-memory reconsolidation. tered during the putative time of cocaine-memory reconsolidation would attenuate subsequent drug context-induced cocaine-seeking behavior. One day after the seventh MATERIALS AND METHODS extinction-training session, rats were re-exposed to the Animals previously cocaine-paired context for 15 min (ie, cocaine- memory reactivation; see experimental timeline in Figure 1a) Male Sprague–Dawley rats (N = 112; 275–300 g; Charles to trigger the destabilization and reconsolidation of context– River Laboratories, Wilmington, MA or Simonsen Labora- response–cocaine memories (Fuchs et al, 2009). The number tories, Gilroy, CA) were individually housed in a tempera- of extinction training sessions was uniform to control for ture- and humidity-controlled vivarium under a reversed memory age, a strong predictor of a memory’s sensitivity to light–dark cycle. Rats were fed 20–25 g of rat chow daily. destabilization (Suzuki et al, 2004; Inda et al, 2011), at the Water was available ad libitum. The housing and treatment time of memory reactivation. Lever pressing was recorded of rats followed the Guide for the Care and Use of Laboratory but was not reinforced during the memory reactivation Animals (National Research Council, 2011) and were session to prevent possible acute cocaine-induced potentia- approved by the IACUC of the University of North Carolina tion of SFK-mediated GluN2A subunit phosphorylation (as Chapel Hill and Washington State University. reported in the ventral tegmental area; Schumann et al, 2009) that is independent of memory reactivation. Immediately Food Training and Surgery after this session, rats received bilateral microinfusions of 0.1% DMSO VEH or PP2 (62.5 ng per 0.5 μl per hemisphere) To expedite cocaine self-administration, rats were first trained into the DH (see Supplementary Methods). This intra-DH to lever press for 45 mg food pellets (Noyes, Lancaster, NH) PP2 dose was selected because it attenuates the expression of during an overnight session in sound-attenuated operant drug context-induced cocaine-seeking behavior (Xie et al, conditioning chambers (Coulbourn Instruments, Allentown, 2013). Treatment assignment was counterbalanced based on PA). Forty-eight hours later, rats were surgically implanted cocaine history. with intravenous jugular catheters and with 26-gauge stain- Beginning the following day, rats received additional daily less-steel guide cannulae (Plastics One, Roanoke, VA) aimed 2 h extinction sessions until they reached the extinction bilaterally at the DH (angled by 15°; AP − 3.4, ML ± 3.1, and criterion (ie, ⩽ 25 active lever responses per session on ⩾ 2 DV − 2.15 mm) or the adjacent trunk region of the somato- consecutive days). Assessment of lever pressing in the sensory cortex (SStr; angled by 15°; AP − 3.4, ML ± 3.1, and extinction context can reveal the possible nonspecific effects DV − 0.65 mm). Surgical and postoperative procedures are of manipulations on general activity and memory (eg, further described in Supplementary Methods. context discrimination impairment because of general

Neuropsychopharmacology SFKs in cocaine-memory reconsolidation AM Wells et al 677

Figure 1 Inhibition of Src family of tyrosine kinases (SFKs) in the dorsal hippocampus (DH) following cocaine-memory reactivation attenuates subsequent drug context-induced cocaine seeking. (a) Schematic depicting the timeline for Experiment 1a. Cocaine self-administration (SA) sessions took place in a distinct environmental context until rats reached the acquisition criterion (* ⩾ 10 infusions per session during ⩾ 10 sessions). Extinction training occurred in a different environmental context. Next, rats were re-exposed to the cocaine-paired (COC-paired) context (cocaine-memory reactivation) for 15 min to trigger cocaine memory destabilization and subsequent reconsolidation. Immediately after the session, rats received bilateral microinfusions of PP2 (a nonselective SFK inhibitor; 62.5 ng per 0.5 μl per hemisphere) or 0.1% dimethyl sulfoxide (DMSO) vehicle (VEH; 0.5 μl per hemisphere) into the DH. Next, the groups received additional extinction training until they met the extinction criterion (# ⩽ 25 non-reinforced active lever responses per session during ⩾ 2 consecutive sessions). Cocaine seeking (non-reinforced active lever responding) was then assessed in the COC-paired context. (b) Photomicrograph and schematics depicting cannula placement. The symbols denote the most ventral point of the injector cannula tracks for rats that received bilateral VEH (n = 6, white circles) or PP2 (n = 8, black circles) infusions into the DH following cocaine memory reactivation in Experiment 1a. Numbers indicate the distance from bregma (in mm), according to the rat brain atlas of Paxinos and Watson (1997). (c) Active and (c′) inactive lever responses during SA (mean ± SEM per 2 h session, average of the last three training sessions), during the cocaine-memory reactivation session (MEM REACT; mean ± SEM per 15 min session), in the extinction context (EXT; mean ± SEM per 2 h session, the last session before the test in the COC-paired context), and in the COC-paired context (mean ± SEM per 2 h session) † for rats in Experiment 1a. (d) Active and (d′) inactive lever responses in 20-min intervals during the test session in the COC-paired context. Significant ‡ difference relative to responding in the extinction context (analysis of variance (ANOVA) context simple main effect, Po0.05). Significant difference relative to VEH treatment (c, ANOVA treatment simple main effect, Po0.05; d, ANOVA treatment main effect, Po0.05). amnesia). Furthermore, the extinction criterion results in (Nader et al, 2000b). The experimental procedures and uniform memory age at test because most rats reach timeline (Figure 2a) followed those in Experiment 1a, except the criterion in 2 days (2.14 ± 0.14 days in the present study) that the rats were exposed to an unpaired context (Context C; and permits demonstration of statistically significant see Supplementary Table S1), which differed from Contexts A drug context-induced reinstatement of drug-seeking and B, for 15 min before receiving bilateral microinfusions of behavior at test by serving as reference. Twenty-four hours VEH or PP2 into the DH. Exposure to the unpaired context after the last extinction session, rats were placed into was expected to provide a similar behavioral experience to that the cocaine-paired context for a 1-h test of drug context- of the cocaine-memory reactivation session without explicit induced cocaine seeking, during which non-reinforced active cocaine-memory reactivation. lever presses were measured as an index of motivation for cocaine. Experiment 2

Experiment 1b In Experiment 2, we evaluated the effects of cocaine-memory reactivation and PP2 treatment on the phosphorylation of In Experiment 1b, we tested whether the effects of PP2 was SFK substrates: GluN2A (Taniguchi et al, 2009) and GluN2B dependent on explicit cocaine-memory reactivation, consis- NMDAR subunits (Nakazawa et al, 2001) and GluA2 tent with a genuine memory reconsolidation impairment AMPAR subunits (Hayashi et al, 1999). We hypothesized

Neuropsychopharmacology SFKs in cocaine-memory reconsolidation AM Wells et al 678

Figure 2 Inhibition of Src family of tyrosine kinases (SFKs) in the dorsal hippocampus (DH) in the absence of explicit cocaine-memory reactivation fails to alter subsequent drug context-induced cocaine seeking. (a) Schematic depicting the timeline for Experiment 1b. Experimental parameters were identical to those in Experiment 1a, except that rats in Experiment 1b were placed into an unpaired context (No memory reactivation) for 15 min before receiving bilateral microinfusions of PP2 (a nonselective SFK inhibitor; 62.5 ng per 0.5 μl per hemisphere) or vehicle (VEH) (0.5 μl per hemisphere) into the DH. (b) Schematics depicting cannula placement. The symbols denote the most ventral point of the injector cannula tracks for rats that received bilateral VEH (n = 7, white circles) or PP2 (n = 7, black circles) infusions into the DH following unpaired context exposure in Experiment 1b. Numbers indicate the distance from bregma (in mm), according to the rat brain atlas of Paxinos and Watson (1997). (c) Mean active lever responses during SA (mean ± SEM per 2 h session, average of the last three training sessions), during unpaired context exposure (NO MEM REACT; mean ± SEM per 15 min session), in the extinction context (EXT; mean ± SEM per 2 h session, the last session before the test in the COC-paired context), and in the COC-paired context (mean ± SEM per 2 h session) † for rats in Experiment 1b. (c′) Inactive lever responses in Experiment 1b. Significant difference relative to responding in the extinction context (analysis of variance (ANOVA) context main effect, Po0.05).

that cocaine-memory reconsolidation would increase Glu- included the approximate volume of PP2 diffusion around N2A subunit phosphorylation and this would be attenuated the injector needle and contained parts of CA1, CA3, and the by PP2. The experimental parameters and timeline in dentate gyrus. Tissue storage and preparation are described Experiment 2 followed those in Experiments 1a, except that in Supplementary Methods. Thirty micrograms of protein rats were exposed to the cocaine-paired context or the home from each sample were electrophoresed on a 7.5% Tris-HCl cage before intra-DH VEH or PP2 microinfusions polyacrylamide gel and transferred to a polyvinylidene (Figure 3a). The home cage condition provides an index of difluoride membrane for 1 h at 100 V. Membranes were basal SFK substrate phosphorylation. Rats were killed 15 min blocked in 5% nonfat milk or Odyssey blocking buffer (LI- or 1 h following intracranial microinfusions to capture a COR Biotechnology, Lincoln, NE) for 1 h and incubated in range of possible phosphorylation kinetics of SFK substrates polyclonal antibodies developed in rabbit against phosphory- (Hayashi and Huganir, 2004; Schumann et al, 2008, 2009; Xie lated GluN2A (pTyr1325), GluN2B (pTyr1472), or GluA2 et al, 2013) related to memory reconsolidation per se (pTyr876) (1:1000) each overnight (16–20 h) at 4 °C. (Pedreira and Maldonado, 2003). Following rapid decapita- Membranes were then incubated in anti-rabbit horseradish tion, brains were removed, flash frozen in isopentane, and peroxidase-conjugated (1:10 000) or infrared secondary stored at − 80 °C. antibody (1:20 000) for 1 h, followed by development with enhanced chemiluminescence (Pierce Biotech, Rockford, IL) Western Blotting or digital infrared imaging systems (LI-COR). Membranes were incubated with stripping buffer (62.5 mM Tris-HCl at Tissue punches of the DH or the SStr were taken using 19 Ga pH 6.7, 2% SDS, 100 mM β-mercaptoethanol) and then neuropunches (Fine Science Tools) from 40 μm tissue reprobed with antibodies developed in rabbit against total sections, collected to verify cannula placement. DH punches GluN2A or GluN2B (1:1000 each), antibodies developed in

Neuropsychopharmacology SFKs in cocaine-memory reconsolidation AM Wells et al 679

Figure 3 Effects of cocaine-memory reactivation and Src family of tyrosine kinase (SFK) inhibition in the dorsal hippocampus (DH) on GluN2A subunit phosphorylation. (a) Schematic depicting the timeline for Experiment 2. Experimental parameters were the same as those used in Experiment 1a, except that rats received bilateral microinfusions of PP2 (62.5 ng per 0.5 μl per hemisphere) or vehicle (VEH) into the DH after re-exposure to the cocaine-paired context (cocaine-memory reactivation) or the home cage (no memory reactivation). (b) Representative photomicrograph depicting the location of the tissue punches in the DH and SStr (trunk region of the somatosensory cortex) and schematics depicting cannula placement for rats that were killed 15 min after receiving cocaine-memory reactivation plus bilateral VEH (15 min: n = 4, open circles) or PP2 (15 min: n = 8, filled circles) infusions into the DH or following exposure to the home-cage plus bilateral VEH (15 min: n = 6, open squares) or PP2 (15 min: n = 6, filled squares) infusions. Numbers indicate the distance from bregma (in mm), according to the rat brain atlas of Paxinos and Watson (1997). (c) Western blot analysis results show mean ( ± SEM) GluN2A phosphorylation (phosphorylated per total protein levels normalized to actin, expressed as percentage of the comparator group (home cage VEH)) in the DH in rats killed 15 min after exposure to the cocaine-paired context (cocaine-memory reactivation) or home cage (no memory reactivation) plus VEH or PP2 treatment. † ‡ Significant difference relative to home cage (analysis of variance (ANOVA) context simple main effect, Po0.05). Significant difference relative to VEH treatment (ANOVA treatment simple main effect, Po0.05). mouse against total GluA2 (1:1000), and later actin (loading parameters and timeline (Figure 4a) followed those in control; 1:25 000). Manufacturer, catalog, and lot information Experiment 1a, except that rats received bilateral microinfu- for each antibody is provided in Supplementary Methods. sions of phosphate-buffered saline VEH or the GluN2A Protein levels were quantified by densitometry, using NIH subunit-preferring NMDAR antagonist (Frizelle et al, 2006), Image J or Image Studio Version 4.0 software. To evaluate PEAQX tetrasodium hydrate (2.5 μg per 0.5 μl per hemi- changes in protein phosphorylation, phosphorylated protein sphere; Sigma-Aldrich, St Louis, MO), into the DH was normalized to total protein and to actin. Protein immediately after cocaine-memory reactivation. This expression is discussed below in terms of normalized levels PEAQX dose was selected because it attenuates cued fear- expressed as a percentage of the comparator group (ie, % memory restabilization (Milton et al, 2013). A third group home cage VEH group). received bilateral microinfusions of PEAQX into the dorsally adjacent SStr following cocaine-memory reactivation to Experiment 3a assess the anatomical specificity of PEAQX effects.

In Experiment 3a, we examined whether antagonism of Experiment 3b GluN2A subunit-containing NMDARs during cocaine- memory reconsolidation impairs subsequent drug context- In Experiment 3b, we tested whether the effects of intra-DH induced cocaine-seeking behavior. The experimental PEAQX in Experiment 3a required explicit cocaine-memory

Neuropsychopharmacology SFKs in cocaine-memory reconsolidation AM Wells et al 680

Figure 4 Antagonism of GluN2A-containing N-methyl-D-aspartate receptors (NMDARs) in the dorsal hippocampus (DH) following cocaine-memory reactivation impairs subsequent drug context-induced cocaine seeking. (a) Schematic depicting the timeline for Experiment 3a. Experimental parameters were identical to those in Experiment 1a, except that rats received bilateral microinfusions of PEAQX (a GluN2A-preferring NMDAR antagonist; 2.5 μg per 0.5 μlper hemisphere), or phosphate-buffered saline vehicle (VEH; 0.5 μl per hemisphere) into the DH after cocaine-memory reactivation. (b) Schematics depicting cannula placement. The symbols denote the most ventral point of the injector cannula tracks for rats that received bilateral vehicle (VEH; n = 8) or PEAQX (n = 9, black triangles) infusions into the DH following cocaine-memory reactivation in Experiment 3a. Numbers indicate the distance from bregma (in mm), according to the rat brain atlas of Paxinos and Watson (1997). (c) Active and (c′) inactive lever responses during SA (mean ± SEM per 2 h session, average of the last three training sessions), during the cocaine-memory reactivation session (MEM REACT; mean ± SEM per 15 min session), in the extinction context (EXT; mean ± SEM per 2 h session, the last session before the test in the COC-paired context), and in the COC-paired context (mean ± SEM per 2 h session ) for rats in Experiment 3a. (d) † Active and (d′) inactive lever responses in 20-min intervals during the test session in the cocaine-paired context. Significant difference relative to responding in the ‡ extinction context (analysis of variance (ANOVA) context main effect, Po0.05). Significant difference relative to VEH treatment (c, ANOVA treatment simple main effect, Po0.05; d, ANOVA treatment main effect, Po0.05).

reactivation. The experimental parameters and timeline RESULTS (Figure 5a) followed those in Experiment 3a, except that Histology rats received bilateral microinfusions of VEH or PEAQX into the DH after unpaired context exposure. Cannula tracks terminated within the DH proper, which included portions of the CA1, CA3, and dentate gyrus Histology subregions (Figures 1, 2, 3, 4 and 5b). High-power micro- Rats in Experiments 1 and 3 were overdosed with ketamine scopy did not reveal tissue damage extensive cell loss or hydrochloride and xylazine (75 and 5 mg/kg, intravenously gliosis at injection sites. Data from rats with incorrect or 150 and 10 mg/kg, intraperitoneally, respectively, depend- cannula placements were excluded from statistical analysis. ing on catheter patency). Brains were removed, postfixed with 10% formaldehyde solution, and sectioned at 75 μm. Sections were stained using cresyl violet (Kodak, Rochester, Behavioral History NY). Cannula tracks were mapped onto schematics from the There were no pre-existing differences between the rat brain atlas (Paxinos and Watson, 1997). experimental groups in Experiments 1, 2, or 3 in cocaine Data Analysis intake or in active or inactive lever responses during self- administration or extinction training or during the memory Data were analyzed using analyses of variance (ANOVAs) reactivation session. Furthermore, these groups did not differ with Tukey’s post hoc tests or t-tests, where appropriate (see in the number of days required to reach the extinction Supplementary Methods for details). Alpha was set at 0.05. criterion aftertreatment (all Fs or ts ⩽ 1.76, Ps ⩾ 0.10).

Neuropsychopharmacology SFKs in cocaine-memory reconsolidation AM Wells et al 681

Figure 5 Antagonism of GluN2A-containing N-methyl-D-aspartate receptors (NMDARs) in the dorsal hippocampus (DH) without cocaine-memory reactivation has no effect on subsequent drug context-induced cocaine seeking. (a) Schematic depicting the timeline for Experiment 3b. Experimental parameters were identical to those in Experiment 3a, except that rats in Experiment 3b received exposure to the unpaired context (no memory reactivation) for 15 min before receiving bilateral microinfusions of PEAQX (a GluN2A subunit-preferring NMDAR antagonist; 2.5 μg per 0.5 μl per hemisphere) or vehicle (VEH; 0.5 μl per hemisphere) into the DH. (b) Schematics depicting cannula placement. The symbols denote the most ventral point of the injector cannula tracks for rats that received bilateral VEH (n = 8, white circles) or PEAQX (n = 8, black circles) infusions into the DH following unpaired context exposure in Experiment 3b. Numbers indicate the distance from bregma (in mm), according to the rat brain atlas of Paxinos and Watson (1997). (c) Active lever responses during SA (mean ± SEM per 2 h session, average of the last three training sessions), during unpaired context exposure (NO MEM REACT; mean ± SEM per 15 min session), in the extinction context (EXT; mean ± SEM per 2 h session, the last session before the test in the COC-paired context), and in the COC- † paired context (mean ± SEM per 2 h session) for rats in Experiment 3b. (c′) Inactive lever responses in Experiment 3b. Significant difference relative to responding in the extinction context (analysis of variance (ANOVA) context main effect, Po0.05).

SFK Inhibition in the DH Following Cocaine Context not differ between the extinction and cocaine-paired contexts. Re-Exposure or Unpaired Context Exposure Time-course analysis of active lever responding during the test Differentially Alters Subsequent Drug Context-Induced session in the cocaine-paired context confirmed that the PP2- Cocaine Seeking treated group responded less on the active lever compared with Intra-DH microinfusions of PP2 administered following the VEH-treated group, independent of time (Figure 1d; = = re-exposure to the previously cocaine-paired context- (ie, ANOVA treatment main effect only, F(1,12) 5.48, P 0.04; = – cocaine-memory reactivation) attenuated subsequent cocaine- all time main and interaction effects, F(2,24) 0.03 3.04, seeking behavior in a context-dependent manner (Figure 1c; P = 0.07–0.95). = Intra-DH microinfusions of PP2 administered following ANOVA context × treatment interaction, F(1,12) 5.88, = = o P 0.03; context main effect, F(1,12) 25.13, P 0.01; treatment exposure to the unpaired context (ie, absence of explicit = = main effect, F(1,12) 4.89, P 0.05). Exposure to the cocaine-memory reactivation) failed to alter subsequent cocaine-paired context increased active lever responding in cocaine-seeking behavior (Figure 2c). At test, exposure to the group that had received VEH into the DH following the cocaine-paired context significantly increased active lever cocaine-memory reactivation, relative to responding in the responding in both the VEH- and PP2-treated groups, extinction context on the preceding day (Tukey’stest, relative to responding in the extinction context (ANOVA = o Po0.05). The PP2-treated group exhibited less active lever context main effect only, F(1,12) 33.47, P 0.01). Moreover, responding in the cocaine-paired, but not in the extinction, there was no difference between the groups in active lever context, relative to the VEH-treated group (Tukey’stest, responding in either context (all treatment main and o = – = – P 0.05). Active lever responding in the PP2-treated group did interaction effects, F(1,12) 0.25 0.42, P 0.53 0.63).

Neuropsychopharmacology SFKs in cocaine-memory reconsolidation AM Wells et al 682 Effects of Cocaine-Memory Reactivation and SFK behavior relative to VEH (Figure 5c). At test, exposure to the Inhibition in the DH on GluN2A, GluN2B, and GluA2 cocaine-paired context significantly increased active lever Subunit Phosphorylation responding in both VEH- and PEAQX-treated groups, relative to responding in the extinction context (ANOVA context Quantitative western blot analyses indicated that total main effect only, F = 24.22, Po0.01). Moreover, the GluN2A subunit levels did not differ across the groups (all (1,14) o 4 groups did not differ in active lever responding in either Fs 3.53, Ps 0.08) but GluN2A subunit phosphorylation in context (all treatment main and interaction effects, the DH was dependent on context exposure and treatment F = 0.00–0.36, P = 0.56–0.99). (Figure 3c; ANOVA context × treatment interaction, (1,14) = = = F(1,20) 5.54, P 0.03; context main effect, F(1,20) 22.99, = P 0.0001). Re-exposure to the cocaine-paired context Inactive Lever Responding increased GluN2A subunit phosphorylation, relative to home cage exposure, 15 min after treatment (Tukey’s test, Po0.05). Inactive lever responding was low in all experiments. The Moreover, intra-DH PP2 treatment reduced GluN2A subunit groups did not differ as a function of treatment, testing phosphorylation following exposure to the cocaine-paired context, or time (all Fs o3.35, Ps 40.05; Figures 1 and 2c′, context (Tukey’s test, Po0.05), but not the home cage, Figures 4 and 5c′, and Figures 1d′ and 4d′). relative to VEH. However, intra-DH PP2 administered following exposure to the cocaine-paired context failed to alter GluN2A subunit phosphorylation in the dorsally DISCUSSION adjacent SStr 15 min after treatment (Supplementary Table In the present study, we demonstrate that activation of S2; t(10) = 1.17, P = 0.27). hippocampal SFKs and stimulation of GluN2A-containing Neither cocaine context re-exposure nor PP2 treatment NMDARs are required for cocaine-memory reconsolidation altered GluN2A subunit phosphorylation 1 h after treatment and drug context-induced instrumental cocaine-seeking (Supplementary Table S2; Fso3.12, Ps40.09). Similarly, behavior. In Experiment 1a, administration of the SFK neither cocaine context re-exposure nor PP2 treatment inhibitor, PP2, into the DH of rats following 15-min re- altered GluN2B or GluA2 subunit phosphorylation at either exposure to the cocaine-associated context-attenuated drug time point (Supplementary Table S2; all Fso2.36, Ps40.14). context-induced cocaine-seeking behavior ~ 72 h later, relative to VEH (Figure 1c). Postsession PP2 administration GluN2A Subunit-Containing NMDAR Antagonism in the was likely time-locked to the onset of memory reconsolida- DH Following Cocaine Context Re-Exposure or Unpaired tion (Fuchs et al, 2009). Furthermore, while lever pressing Context Exposure Differentially Alters Subsequent Drug was not reinforced during memory reactivation, it is unlikely Context-Induced Cocaine Seeking that postsession PP2 administration enhanced extinction memory consolidation. The 15-min memory reactivation Intra-DH PEAQX administration following cocaine-memory session did not result in observable extinction learning; thus, reactivation attenuated subsequent cocaine-seeking behavior it is improbable that the extinction memory trace was the in a context-dependent manner (Figure 4c; ANOVA dominant memory trace at the onset of memory reconso- = = context × treatment interaction, F(1,15) 5.13, P 0.04; con- lidation (Eisenberg et al, 2003). Moreover, extant literature = o text main effect, F(1,15) 47.00, P 0.01; treatment main indicates that postsession intra-DH PP2 administration = = effect, F(1,15) 6.17, P 0.03). Exposure to the cocaine-paired impairs, rather than enhances, extinction memory consoli- context significantly increased active lever responding in the dation in other experimental procedures (Bevilaqua et al, group that had received VEH into the DH following cocaine- 2005). A similar effect would have been indicated by memory reactivation, relative to responding in the extinction increase, rather than the observed decrease (Figure 1c), in context on the preceding day (Tukey’s test, Po0.05). The active lever pressing at test in our model. Similarly, PP2 PEAQX-treated group exhibited less active lever responding effects cannot be attributed to protracted hypoactivity or in the cocaine-paired, but not in the extinction context, general amnesia, as PP2 administration following unpaired relative to the VEH-treated group (Tukey’s test, Po0.05). context exposure (ie, no memory reactivation) did not Active lever responding in the PEAQX-treated group did not attenuate subsequent drug context-induced cocaine-seeking significantly differ between the two contexts. Time-course behavior, relative to VEH (Figure 2c). PP2 also failed to alter analysis of active lever responding during the test session in active lever responding in the extinction context (Figure 1c) the cocaine-paired context revealed that the PEAQX-treated and inactive lever responding in either context, relative to group exhibited fewer active lever responses than the VEH- VEH (Figure 1c0), albeit these measures are subject to floor treated group, independent of time (Figure 4d; ANOVA effects. Finally, diffusion of PP2 away from the DH to other = = treatment main effect only, F(1,15) 5.73, P 0.03; all time brain regions was negligible. Consistent with this, intra-DH = – = – main and interaction effects, F(2,30) 0.40 1.56, P 0.23 PP2 treatment did not alter GluN2A subunit phosphoryla- 0.68). Furthermore, a third group that had received PEAQX tion in the dorsally adjacent SStr, which falls in the most into the dorsally adjacent SStr (n = 6) exhibited more active probable path of drug diffusion along the cannula track lever responses in the cocaine-paired context (mean ± SEM = (Supplementary Table S2). Overall, these findings suggest 37.33 ± 9.43; data not shown) compared with that in the that PP2 in the DH selectively disrupted the reconsolidation = = extinction context (t(5) 3.07, P 0.03), similar to the VEH- of cocaine memories. treated group. GluN2A NMDAR subunit activation was also necessary Intra-DH PEAQX administration following exposure to the for cocaine-memory reconsolidation (Figure 4c). In Experi- unpaired context failed to alter subsequent cocaine-seeking ment 3a, administration of the GluN2A-preferring

Neuropsychopharmacology SFKs in cocaine-memory reconsolidation AM Wells et al 683 antagonist, PEAQX, into the DH of rats following cocaine- 2005; Yang et al, 2012). Therefore, future studies will be memory reactivation attenuated subsequent drug essential to elucidate the contribution of specific SFKs to context-induced cocaine-seeking behavior, relative to VEH. discrete memory processes (eg, destabilization and reconso- Recapitulating the effects of PP2, PEAQX effects were lidation) involved in the prolonged maintenance of salient dependent on explicit cocaine-memory reactivation, in that and intrusive drug-related memories. PEAQX treatment administered after unpaired context It is noteworthy that neither cocaine-memory reactivation exposure was without effect at test (Figure 5c). PEAQX nor PP2 treatment altered GluN2B or GluA2 subunit effects were also anatomically selective, such that PEAQX activation in the DH (Supplementary Table S2). Fyn- treatment administered into the SStr did not attenuate dependent phosphorylation of GluN2B-containing cocaine seeking at test (data not shown). These findings NMDARs, in particular, has been shown to support extend research demonstrating the involvement of GluN2A- ethanol-seeking behaviors (Wang et al, 2007, 2010) and the containing NMDARs in the BLA in auditory fear-memory context-induced reinstatement of cocaine-seeking behavior reconsolidation (Milton et al, 2013). (Xie et al, 2013). The apparent lack of involvement of SFKs tightly regulate GluN2A NMDAR subunit activation GluN2B subunits in the present study highlights distinct (Taniguchi et al, 2009). Thus, we examined the phosphor- mechanisms governing the expression of relapse-like beha- ylation state of GluN2A subunits, as well as alternate SFK viors and the maintenance of long-term memories subser- substrates—GluN2B NMDAR and GluA2 AMPAR ving this phenomenon. subunits—following cocaine-memory reactivation or no In summary, the present findings point to SFKs and memory reactivation and intra-DH PP2 or VEH treatment. GluN2A-containing NMDARs as novel intracellular me- Cocaine-memory reactivation increased GluN2A subunit chanisms through which the DH may critically support phosphorylation compared with no memory reactivation at drug-memory reconsolidation (Ramirez et al, 2009), perhaps 15 min, but not at 1 h (Supplementary Table S2), post- by supporting protein synthesis-dependent cocaine-memory memory reactivation (Figure 3c). Transient GluN2A activa- restabilization in other brain regions, such as the BLA (Fuchs tion is consistent with the rapid phosphorylation/ et al, 2009; Wells et al, 2011), or by promoting the dephosphorylation kinetics of GluN2A NMDAR subunits establishment of retrieval links to recently restabilized (Vincini et al, 1998). Src-mediated phosphorylation of cocaine memories (Wells et al, 2011). This may require GluN2A subunits promotes NMDAR surface expression direct phosphorylation of GluN2A subunits by Src (Taniguchi et al, 2009), potentiates NMDAR currents in the (Taniguchi et al, 2009), although future studies will be striatum in vivo (Taniguchi et al, 2009), and enhances LTP at necessary to show that this interaction is requisite. Never- CA1 synapses in vitro (Yang et al, 2012). Among others, theless, the present findings begin to clarify the complex role LTP-like processes promote memory reconsolidation (Nader of the DH in cocaine-memory reconsolidation and may et al, 2000a; Alberini, 2006; Abraham and Williams, 2008). inform the development of treatments intended to weaken Thus, the increase in GluN2A phosphorylation following drug-related memories. cocaine-context exposure likely reflects obligatory GluN2A regulation during memory reconsolidation. In support of this hypothesis, intra-DH PP2 treatment administered following FUNDING AND DISCLOSURE exposure to the cocaine-paired context, but not the home This research was funded by NIDA R01 DA025646 (RAF) cage, attenuated both the transient increase in GluN2A and NIDA F31 DA034391 (AMW). The authors declare no subunit activation in the DH (Figure 3c) and subsequent conflict of interest. cocaine-seeking behavior in a separate cohort of rats (Figure 1c and d). 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